Carriers thyroglobulin

Whether the choice of carrier significantly influences the antihapten response is a controversial subject. In the author s opinion, no really definitive study has been carried out. On the other hand, there are some who believe that KLH is a superior carrier. Thyroglobulin is a choice of others.As noted above, we chose the serum albumins, as have the great majority of the laboratories engaged in developing radioimmunoassays. 

The most common carrier proteins in use today are keyhole limpet hemocyanin (KLH MW 4.5 X 105 to 1.3 X 107), BSA (MW 67,000), aminoethylated (or cationized) BSA (cBSA), thyroglobulin (MW 660,000), ovalbumin (OVA MW 43,000), and various toxoid proteins, including tetanus toxoid and diphtheria toxoid. Other proteins occasionally used include myoglobin, rabbit serum albumin, immunoglobulin molecules (particularly IgG) from bovine or mouse sera, tuberculin purified protein derivative, and synthetic polypeptides such as poly-L-lysine and poly-L-glutamic acid. 

Thyroglobulin and ovalbumin (OVA) are used less often as carriers, but they are particularly valuable as non-relevant carriers in ELISA tests designed to measure the antibody response... 

Since OVA and BSA possess some immunologically similar epitopes, a population of the antibodies produced against one often will cross-react against the other. Therefore, OVA cannot function as a non-relevant carrier for BSA and vice versa. Either OVA or BSA, however, may be used as non-relevant carriers for KLH, thyroglobulin, or the various toxoid proteins used as immunogen conjugates. 

To become immunogenic, a hapten has to be linked with a large molecule, such as a protein, prior to its introduction into the host animal. Several proteins can act as such carrier molecules including bovine serum albumin, human serum albumin, ovalbumin, thyroglobulin, poly-L-lysine, and hemocyanin. Among these proteins, bovine serum albumin is most commonly used because it is inexpensive, readily available, very soluble, highly immunogenic, and, in addition, resists denaturatlon (3). 

Weigh out the peptide and an equal weight of KLH or thyroglobulin carrier. This gives an approximate ratio of 40-150 molecules of peptide to each molecule of carrier (2 mg of peptide per animal is ample). If... 

TVansport—proteins may act as carriers to transport other molecules. For instance, hemoglobin is a protein that carries Oj in red blood cells myoglobin transports O, in muscle cells transferrin transports iron thyroglobulin-binding protein transports thyroxin. Albumin forms the largest proportion of plasma protein. It carries various hormones, iron, heme, vitamins, bilirubin, frr f" sc-... 

No definitive study of the effects of protein carrier on response is available, and many carrier proteins have been used, including globulins, albumin, hemocyanin, thyroglobulin, and fibrinogen. The optimal number of haptens, or the epitope density, is also controversial, but a density of 8 to 25 haptens per bovine serum albumin molecule is probably optimal (12),... 

The choice of the carrier is important. The most common carriers are serum albumin of various species (generally quite soluble Erlang-er et al, 1959), keyhole limpet hemocyanin, thyroglobulin, ovalbumin, or fibrinogen. Different carriers can be used for immunization and assays or, alternatively, antibodies to the carrier can be removed by absorption. 

Fig. 5 Immobilization of analyte-protein conjugate. A small molecule can be conjugated to a carrier protein such as BSA or thyroglobulin and be immobilized onto a solid phase antigen-protein-solid phase (Ag-P-SP). Labeled primary or secondary antibody (AbE) and sample (Ag) are introduced. The analyte in the sample competes with the immobilized Ag to form immunocomplex (Ab-Ag or AbE-Ab-Ag-P-SP) with the limited amount of antibody. Ab-Ag is washed away, and the AbE that is not occupied by the sample analyte is detected by substrate signal development.

The protein carriers used in various laboratories include globulin fractions, serum albumins of different species, hemocyanin, ovalbumin, thyroglobulin, and fibrinogen. Also nonproteinaceous carriers have been used... 

There has been much speculation in the past on the nature of the circulating thyroid hormone whether it was a simple amino acid, a peptide or polypeptide of thyroxine, or thyroglobulin itself. In 1948 Taurog and Chaikoff produced a considerable amount of evidence that the plasma hormone was indeed thyroxine. Labeled plasma iodine behaved chemically in a manner identical with that of thyroxine added to plasma it was nondialyzable it was precipitated with plasma proteins with zinc hydroxide it was extractable with butanol, and it could be fractionated with carrier thyroxine by its partition between immiscible solvents. This work received confirmation from Laidlaw (1949), and it then became generally accepted that thyroxine alone was the circulating hormone. 

The method generally used for the preparation of the bile acid-protein conjugate is "mixed anhydride" technique described by Erlanger et al. or the carbodiimide method. Some authors have used thyroglobulin instead of bovine serum albumin as carrier protein of the immunogen because of the higher molecular weight. 

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